Short hairpin RNA (shRNA) knockdown reagents
The RNA Interference Core provides access to lentiviral short hairpin ribonucleic acid (shRNA) knockdown vectors against human and mouse genes. Reagents are provided as bacterial stocks. Both Sigma-Aldrich and Open Biosystems produce target sets, with multiple constructs targeting a given gene. Within the Sigma-Aldrich Mission shRNA library, each target set consists of three to five constructs, with an average of 4.9 constructs per set. The Open Biosystems library is less consistent, with 10 percent of targets being represented by only one construct, others being represented by as many as 10 constructs and an average of 2.4 clones per set.
In either case, constructs within a target set must be screened to identify the optimum knockdown reagents. Plasmid maxipreps must be made from all three to five constructs and used to transfect HEK293-based packaging cell lines, obtained from Invitrogen as part of the ViraPower kit. Culture supernatants from these packaging cells may then be used for transient transduction of the cell line of interest.
Ribonucleic acid is harvested 48 hours after infection, and the efficiency of knockdown is assessed by quantitative real-time polymerase chain reaction. Western blotting may be used if well-characterized antibodies are available.
Inducible shRNA knockdown reagents
The Sigma-Aldrich Mission group has recently made available an isopropyl beta-D-thiogalactopyranoside (IPTG) inducible vector backbone, into which hairpin sequences from traditional shRNA constructs can be cloned. This service is thus far offered only as a custom feature, but it is possible (and recommended by the RNA Interference Core) that investigators choose constructs from the available Mission shRNA knockdown library that they have already characterized to insert into the new inducible backbone.